NOTES
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Vol. 77
also included in Table I. The poor precision of the involved the removal of an acidic hydrogen from results plus the fact that the value of the distribu- the reagent. Similar coordination compounds of tion coefficient differed so markedly from that for palladium6s7have been reported. uranyl ion leads to the hypothesis that a reaction Experimental other than the exchange is taking place. Despite Materials.--A weighed amount of palladium(I1) chloride, the fact that Np(V1) is stable in molar perchloric obtained from Coleman and Bell Company, was dissolved acid with respect to reduction to Np(V) the obvious in a small amount of concentrated hydrochloric acid and assumption to make is that the organic substrate, then diluted t o volume with distilled water. The resulting solution, which was approximately 0.1 M in hydrochloric i.e., either the hydrocarbon skeleton of the Dowex- acid, was analyzed for palladium content both gravimetri50 and/or the nuclear sulfonic acid which acts as callye and polarographically.9 Standard 1,2,3-benzotriazoIe solution in 50% acetic acid the exchange group, promotes this reduction. This conclusion is supported by the data included in Ta- was prepared after recrystallizing this reagent, Eastman Kodak Chemical Aye. 2759, twice from chloroform; the ble I for the distribution of Np(V) between molar melting point of the recrystallized reagent was 100.0”. perchloric acid and the resin.6 All other materials used were reagent grade chemicals. Apparatus.-A mechanized, micro-combustion Sargen t The reduction of Xp(V1) to Np(V) was then qualitatively demonstrated by equilibrating a solu- apparatus was employed for the determination of carbon, and palladium. tion of 6 X lop3 M Np(V1) in 0.955 M perchloric hydrogen Procedure.-To four beakers each containing 0.417 acid with about 0.2 g. of the dry resin for a period mmole of palladium were added 10 ml. of 2 M acetic acidof one hour. At the end of this time the solution sodium acetate buffer, and a slight excess of 1,2,3-benzowas centrifuged and an absorption spectra of the triazole (dissolved in 50% acetic acid); one palladium ion in excess benzotriazole reacts with two molecules of the reaqueous layer was taken. The spectra showed the agent. The white colored precipitates were digested for 10 presence of Np(V). Within the experimental un- minutes, and then filtered using weighed, medium porosity, certainties reduction was complete. The slow in- sintered-glass crucibles. The precipitates were washed crease in the distribution coefficient in the Np(V) several times with dilute hydrochloric acid (1 :loo), and finally several times with distilled water. After each presolution indicates a further reduction to Np(1V) is cipitate had been dried at 110’ for 1 hour to constant weight, occurring. the following weights of the four precipitates were obtained: In the light of these observations the correlation 174.1, 173.0, 173.4, 173.0 mg. Anal. Calcd. for the white between cation exchange column behavior and the colored precipitate: C, 34.66; H, 2.42; C1, 17.06; N, Pd, 25.66. Found: C, 34.68; H, 2.45; C1, 17.05; degree of association between Np(V1) and chloride 20.21; S, 20.19; Pd, 25.64. ion7is subject to reservations. Aliquots of the standard solution of palladium were (6) Qualitative tests for t h e presence of C1- and iron in t h e wash liquid from t h e resin were negative. Resin t h a t had been equilibrated f o r as long as 24 hours with molar perchloric acid showed some evidence of decomposition, namely, a positive test for sulfate in t h e aqueous phase. (7) R . M. Diamond, K , Street, Jr., and G. T. Seaborg, THISJOURK A L , 76, 1461 (1954).
CHEMISTRY DIVISION h TLABORATORY ~ ~ ARGONNE ILLISOIS LEMONT,
~
~
Preparation of Palladium(I1) Chloride-l,2,3-Benzotriazole Coordination Compounds‘ BY RAYF.WILSONA N D LOUBERTA E. WILSON RECEIVEDJULY 11, 1955
transferred t o three beakers, and 10 ml. of buffer added to each beaker. Then standard 1,2,3-benzotriazole solution was added in amounts such that the molar concentration of palladium remained in slight excess; in each case 0.500 mmole of reagent was added. After standing a t room temperature for 12 hours, the precipitates were filtered using fine porosity, sintered-glass crucibles and were washed, dried and weighed in the usual manner. The three reddishbrown precipitates gave the following weights: 148.1, ~ and 148.1 ~ 147.6 mg.~ Anal. Calcd. for the reddishbrown colored precipitate: C, 24.28; H, 1.70; Cl, 23.90; N, 14.16; P d , 35.96. Found: C, 24.29; €1, 1.70; C1, 23.89; iY,14.14; Pd, 38.96. Quantitative analysis for nitrogen was carried out using micro Kjeldahl procedureLo for chlorine by the Carius method’o; and for carbon, hydrogen and palladium by modifying slightly the procedure described by Steyermark.lo X Sargent carbon-hydrogen apparatus was employed for the micro-determination of carbon and hydrogen. Following the determination, the carbon dioxide and the mater absorption tubes were removed from the apparatus, and the palladium was ignited again in an atmosphere of carbon dioxide to make sure that all the palladium and/or palladium residue had been converted to the metal.
In connection with a general study of the reactions between the platinum elements and 1,2,3-benzotriazole, two palladium coordination compounds have been prepared. 1,2,3-Benzotriazole was first suggested as a precipitant for silver by Remington Discussion and Moyer2 and later by Tarasevich3 and C h e ~ ~ g . ~Palladiuni(I1) chloride reacts with 1,2,3-benzoCurtis5 has employed the same reagent for the de- triazole to form two different coordination comtermination of copper. These authors report that pounds, depending on which is in excess, palladium nickel(IT), cobalt(II), iron(II), zinc and cadmium or 1,2,3-benzotriazole. Both the white and the are also precipitated by 1,2,3-benzotriazole; how- reddish-brown colored precipitates were very inever, a study of the interaction of palladium or other soluble in most organic solvents and in most conplatinum elements with this reagent appears not to centrated inorganic acids. These precipitates aphave been made previously. The reactions of pre- peared to be sparingly soluble in warm sulfuric acid ; viously reported ions with 1,2,3-benzotriazole, in (6) J. €I. Y o e and L. 0. Overholser, Tms J O U R N A L , 61,2059 ( 1 9 3 9 ) . contrast to the corresponding palladium reactions, (7) J. R. Hayes and G. C. Chandler, Ind. E n g . Chcm., Anal. Ed., 14, 11) This work was supported b y a grant from t h e National Science Foundation. ( 2 ) W , J . Remington and H. 1‘. Moyer, “Dissertation Abstract,” Ohio State Univ. Press, Columbus. Ohio, 1937, p. 24. (3) N.I. Tarasevich, Veslnik Moskov. Unio., 3 , No. 10, 161 (1948). (4) K. L . Cheng, Anol. Chem., 26, 1038 (19.54). (.i) J . A . Curtis I d . E i t g . Cheni., Anal. E d . , 13, 349 (1941).
491 (1942). (8) W. F. Hillebrand, G. E. F. Lundell, H. A. Bright und J. I . Hoffman, “Applied Inorganic Analysis,” 2nd ed., John Wiley and Sons, Inc., New York, N. Y . , 1953, p. 379. (9) R . F. Wilson and R . C. Daniels, Anal. Chem., 27, 904 (1955). (10) A. Steyermark, “Quantitative Organic hlicroanalysis,” T h e Blakiston Company, Philadelphia, Pa., 1051, pp. 82-191.
Dec. 5, 1955
NOTES
the dried precipitates were stable up to temperatures of a t least 300”. Weight relations under “Experimental” and the elemental analyses gave convincing evidence for the formation of two coordination type compounds in the interaction of palladium(I1) chloride and 1,2,3-benzotriazole. On the basis of the palladium content, the observed formulas are Pd(CsHrNHN&ClZ and Pd(C6H4NHNZ)ClZ,and their formula weights are 415.86 and 296.74, respectively.
mechanism will be referred to as the carbon dioxide mechanism. It is well-known that a t room temperature there
DEPARTMENT O F CHEMISTRY TEXAS SOUTHERN UNIVERSITY 4,TEXAS HOUSTON
On the Mechanism of Urease Action BY Jur H. WANGAND DONALD A. TARR RECEIVED JULY 18, 1955
There are three proposed mechanisms for the hydrolysis of urea by urease in slightly acid solutions.’ First we have the carbonic acid mechanism in which urea is assumed to be hydrolyzed first to carbamic acid, then to carbonic acid, and finally to carbon dioxide and water. Secondly we have the carbamic acid mechanism in which urea is first hydrolyzed to carbamic acid, and then the latter decomposes directly to carbon dioxide and ammonia without going through the carbonic acid stage. The weight of indirect experimental evidences in the literature seems to favor this latter mechanism. However, Sumner and Somersl recently proposed a third mechanism in which urea is directly hydrolyzed to carbon dioxide and ammonia without even going through the carbamic acid stage. These authors believe that the formation of ammonium carbamate in the absence of buffers is due to the recombination of the primary products COz and ”3. But i t is by no means apparent just how urea can be hydrolyzed to COz and NH3 without going through the carbamic acid or carbamate stage. One possible mode of action is the reaction between a water molecule and a urea molecule under the influence of urease such that the two amide groups are simultaneously broken off and replaced by a single oxygen atom from the water molecule. But such a mechanism would require the simultaneous rupture of four bonds (two N -C bonds of urea and two H-0 bonds of water) with the concurrent formation of three new ones (two H-N bonds of two ammonia molecules and one C=O bond of the carbon dioxide molecule), and is hence extremely unlikely. Another possible mechanism for the direct production of COZ is the replacement of the oxygen in urea by an unknown basic group on the urease molecule, the two amide groups are then hydrolyzed off one after the other, and finally the COz is detached from the enzyme. In this way urea would indeed be converted to carbon dioxide and ammonia without going through the carbamate stage. Although there is no direct evidence to support this last mechanism, i t would be risky to exclude it from our consideration in view of Sumner and Somers’ suggestion. I n the following discussion, this last (1) For references t o t h e literature see J. B. Sumner a n d G. F. Somers, “Chemistry and Methods of Enzymes,’’ Academic Press, Inc., New York, N . Y . , 1953, pp. 157-158,
6205
is no detectable 0I8-exchange between urea and water,2 and that the OI8-exchangebetween carbon dioxide and water is also slow in the absence of carbonic a n h y d r a ~ e . ~Consequently if ordinary urea is rapidly hydrolyzed by urease in slightly acid, Hz018-enrichedwater solution and the carbon dioxide liberated is immediately separated from the liquid mixture for mass-spectrometric analysis, the result should enable us to decide which one of the three mechanisms described above is correct. For convenience of the subsequent discussion, let us introduce the new term “enrichment ratio” defined below. Enrichment ratio = atom % excess of 0 1 8 in COZjust liberated from the urea soln. atom % excess of 0l8in COS equilibrated with the urea soln.
The quantity in the denominator of the above ratio is determined by keeping the liberated COZ in contact with the liquid mixture for more than 5 hr., then separating it from the liquid and analyzing for 018-content. A moment’s consideration should convince us that the three mechanisms of urease action described above predict different enrichment ratios. The carbonic acid mechanism predicts an enrichment ratio of 2/3, the carbamic acid mechanism predicts a ratio of I/z, and the carbon dioxide mechanism predicts a ratio of unity. Water containing about 1.5 atom yo of 0l8was used as tracer in this work. The results are summarized in Table I. Three kinds of measurements were carried out. First dry sodium bicarbonate powder was mixed with excess of HzO18-enriched soln. of urea in citrate buffer ($H 5.5) in an evacuated vessel for ‘/zminute a t 0”. The mixture was then chilled in a Dry Ice-acetone bath, and the liberated COz was sampled out for mass spectrometric analysis. The results of two such measurements listed in Table I (expts. 8 and 9) give an average 0 I 8 atom % of 0.22. Since the natural abundance of 0ls is about 0.20 atom ’% and the water in the buffer s o h . contained about 1.5 atom % in OIs, it is clear that under these experimental conditions the rate of 018-exchangebetween liberated COZand water is relatively slow and may be considered as negligible in all the other l/Z-minute experiments a t 0” described below. In the second group of measurements the HZOI8enriched soln. of urea in citrate buffer was mixed with equal volume of H2018-enrichedsoln. of urease in the same buffer for 1/2 min. a t 0”. The liberated C02 was separated and analyzed as before. The results of three determinations listed in Table I (expts. 1, 2 and 3) give an average 0l8atom % of 0.863. I n the last group of measurements, the same urea and urease soln. were mixed as before, but the liberated COz was left in contact with the H2018-enriched soln. for several hr. to reach exchange equilibrium before mass spectrometric analysis. The average 0l8atom % in the equilibrated COz obtained from four determinations (expts. 4,5, 6 and 7 in Table I) is 1.47. Thus the (2) M. Cohn and H. C. Urey, THISJOURNAL, 60, 679 (1938). ( 3 ) H. C . Urey and L. J . Greiff, ibid.. 57, 321 (1936).
